Organoarsenic compounds and methods for the treatment of cancer

ABSTRACT

A method for treating a lymphoma selected from non-Hodgkin&#39;s and Hodgkin&#39;s lymphoma comprising administering an organoarsenic compound having a structure of the formula (I) wherein X is S or Se and R 1  and R 2  are independently C 1-30 alkyl (R 3 , R 3′ , R 4 , R 5 , W and “n” are as defined in claim  1 ) in particular where the compound is S-dimethylarsinoglutathione, N-(2-S-dimethylarsinothiopropionyl)glycine, 2-amino-3-(dimethylarsino)thio-3-methylbutanoic acid, S-dimethylarsino-thiosuccinic acid or S-dipropylarsino-1-thioglycerol.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/189,511 filed Aug. 20, 2008, which is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of anti-cancertherapy. More particularly, it provides organic arsenic compounds andmethods for their use in treating cancers such as leukemia and solidtumors.

BACKGROUND OF THE INVENTION

Despite progress in leukemia therapy, most adult patients with leukemiastill die from disease progression. Arsenic trioxide, an inorganiccompound, has been approved for the treatment of patients with relapsedor refractory acute promyelocytic leukemia (APL) and is being evaluatedas therapy for other leukemia types. Preliminary data from China and therecent experience in the U.S., however, suggest a role for arsenictrioxide in the other hematologic cancers as well. Consequently, theactivity of arsenic trioxide as an anti-leukemic agent is currentlybeing investigated in many types of leukemia. Although the results lookfavorable in terms of the response rate of some of the leukemia typesthat are being investigated, systemic toxicity of arsenic trioxide is aproblem (Soignet et al., 1999; Wiernik et al., 1999; Geissler et al.,1999; Rousselot et al., 1999).

The only organic arsenical (OA) manufactured for human use, melarsoprol,has been evaluated for antileukemic activity (WO9924029, EP1002537).Unfortunately, this compound is excessively toxic to patients withleukemia at concentrations used for the treatment of trypanosomiasis.Therefore, there is a need to identify arsenic derivatives that can beused for the treatment of hematologic malignancies and cancer ingeneral, that have similar or greater activity and lower toxicity thanarsenic trioxide.

SUMMARY OF THE INVENTION

The present invention provides organic arsenical compounds withanti-cancer properties. In some embodiments, the present inventionprovides compounds having a structure of formula (I) or apharmaceutically acceptable salt thereof

-   wherein-   X is S or Se;-   W is O, S, or (R)(R), where each occurrence of R is independently H    or C₁₋₂alkyl;-   n is an integer from 0 to 20;-   R¹ and R² are independently C₁₋₃₀alkyl;-   R³ is —H, C₁₋₁₀alkyl, or C₀₋₆alkyl-COOR⁶;-   R^(3′) is H, amino, cyano, halogen, aryl, aralkyl, heteroaryl,    heteroaralkyl, carboxyl, C₁₋₁₀alkyl, C₁₋₁₀alkenyl, or C₁₋₁₀alkynyl,    preferably H;-   R⁴ is —OH, —H, —CH₃, amino, —OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl,    —OC(O)aryl, or a glutamine substituent;-   R⁵ is —OH, cyano, C₁₋₁₀alkoxy, amino, O-aralkyl, —OC(O)C₁₋₁₀aralkyl,    —OC(O)C₁₋₁₀alkyl, —OC(O)aryl, or a glycine substituent; and-   R⁶ is H or C₁₋₁₀alkyl.

In certain embodiments, the organic arsenicals are compounds having astructure of formula (II)

-   wherein-   X is S or Se, preferably S;-   W is O, S, or (R)(R), where each occurrence of R is independently H    or a C₁₋₂alkyl, preferably O;-   Z is CH or N, preferably N;-   R¹ and R² are independently C₁₋₁₀alkyl, preferably R¹ and R² are    independently selected from methyl, ethyl, propyl, and isopropyl;    and-   R⁵ is —OH, cyano, C₁₋₁₀alkoxy, amino, O-aralkyl, O-aralkyl,    —OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl, —OC(O)aryl, or a glycine    substituent, preferably OH;-   R⁶ is H or C₁₋₁₀alkyl;-   R⁷ is selected from halogen, —OH, C₀₋₆alkyl-COOR⁶, C₁₋₆alkyl,    C₁₋₆alkoxy, amino, amido, cyano, and nitro;-   m is an integer from 0 to 4, preferably 0.

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a number of organic arsenic compounds.

In certain embodiments, the organic arsenicals of the present inventionhave a structure of formula (I) or a pharmaceutically acceptable saltthereof

-   wherein-   X is S or Se, preferably S;-   W is O, S, or (R)(R), where each occurrence of R is independently H    or a C₁₋₂alkyl, preferably O or (R)(R);-   n is an integer from 0 to 20;-   R¹ and R² are independently C₁₋₃₀alkyl;-   R³ is —H, C₁₋₁₀alkyl, or C₀₋₆alkyl-COOR⁶;-   R^(3′) is H, amino, cyano, halogen, aryl, aralkyl, heteroaryl,    heteroaralkyl, carboxyl, C₁₋₁₀alkyl, C₁₋₁₀alkenyl, or C₁₋₁₀alkynyl,    preferably H;-   R⁴ is —OH, —H, —CH₃, amino, —OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl,    —OC(O)aryl, or a glutamine substituent;-   R⁵ is —OH, cyano, C₁₋₁₀alkoxy, amino, O-aralkyl, —OC(O)C₁₋₁₀aralkyl,    —OC(O)C₁₋₁₀alkyl, —OC(O)aryl, or a glycine substituent; and-   R⁶ is H or C₁₋₁₀alkyl, preferably H.

In certain embodiments, W is (R)(R) and each occurrence of R isindependently H or a C₁₋₂alkyl. In certain such embodiments, eachoccurrence of R is H.

In certain embodiments, n is 0 or 1, preferably 1. In certainembodiments, n is an integer from 2 to 20, preferably from 5 to 20 or 9to 14.

In certain embodiments, R¹ and R² are each independently C₁₁₋₃₀alkyl,preferably C₁₂₋₂₈alkyl, C₁₃₋₂₅alkyl, C₁₄₋₂₂alkyl, or even C₁₅₋₂₀alkyl.

In certain embodiments, R¹ and R² are C₁₋₁₀alkyl, preferably R¹ and R²are independently selected from methyl, ethyl, propyl, and isopropyl

In certain embodiments, R³ is —H or C₀₋₆alkyl-COOR⁶. In certain suchembodiments, R³ is selected from —COOR⁶, —CH₂COOR⁶, —CH₂CH₂COOR⁶,—CH(CH₃)COOR⁶, —CH(CH₂CH₃)COOR⁶, or —CH₂CH₂CH₂COOR⁶, wherein R⁶ isC₁₋₁₀alkyl.

In certain embodiments, R³ is C₁₋₁₀alkyl. In certain preferred suchembodiments, R³ is selected from methyl, ethyl, propyl, and isopropyl,preferably methyl.

In certain embodiments, R^(3′) is selected from amino, cyano, halogen,aryl, aralkyl, heteroaryl, heteroaralkyl, carboxyl, C₁₋₁₀alkyl,C₁₋₁₀alkenyl, and C₁₋₁₀alkynyl. In preferred such embodiments, R^(3′) isselected from aryl, aralkyl, heteroaryl, heteroaralkyl, carboxyl,C₁₋₁₀alkenyl, and C₁₋₁₀alkynyl

In certain embodiments, R⁴ is selected from —OH, —H, —CH₃,—OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl, and —OC(O)aryl. In certain suchembodiments, R⁴ is selected from —OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl,and —OC(O)aryl.

In certain embodiments, R⁴ is amino. In certain such embodiments, R⁴ isNH₂.

In certain embodiments, R⁴ is a glutamine substituent.

In certain embodiments, R⁵ is selected from cyano, C₁₋₁₀alkoxy, amino,O-aralkyl, —OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl, and —OC(O)aryl.

In certain embodiments, X is S, W is (R)(R), wherein each occurrence ofR is

H, n is 1, R¹ and R² are independently selected from methyl, ethyl,propyl, and isopropyl, R³ and R^(3′) are H, R⁴ is selected from OH,—OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl, and —OC(O)aryl and, and R⁵ isselected from OH, —OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl, and —OC(O)aryl.In certain such embodiments, R¹ and R² are the same and are togetherselected from methyl, ethyl, propyl, and isopropyl.

In certain embodiments, X is S, W is O, n is 1, R¹ and R² are bothmethyl, R³ is selected from H and COOR⁶, R^(3′) is H, and R⁴ is selectedfrom H and a glutamine substituent, and R⁵ is selected from OH and aglycine substituent. In certain such embodiments, R³ is COOR⁶, R⁴ is H,R⁵ is OH, and R⁶ is H.

In certain embodiments, compounds of formula (I) are selected from

or a pharmaceutically acceptable salt thereof.

In certain embodiments, compounds of formula (I) are selected from

or a pharmaceutically acceptable salt thereof.

In certain embodiments, compounds of formula (I) are selected from

or a pharmaceutically acceptable salt thereof.

In certain embodiments, compounds of formula (I) are selected from

In certain embodiments, a compound of formula (I) is

In certain embodiments, a compound of formula (I) is

or a pharmaceutically acceptable salt thereof.

If a chiral center is present, all isomeric forms are within the scopeof the invention. Regarding the stereochemistry, the Cahn-Ingold-Prelogrules for determining absolute stereochemistry are followed. These rulesare described, for example, in Organic Chemistry, Fox and Whitesell;Jones and Bartlett Publishers, Boston, Mass. (1994); Section 5-6, pp177-178, which section is hereby incorporated by reference.

In certain embodiments, the organic arsenicals are compounds having astructure of formula (II)

-   wherein-   X is S or Se, preferably S;-   W is O, S, or (R)(R), where each occurrence of R is independently H    or a C₁₋₂alkyl, preferably O;-   Z is CH or N;-   R¹ and R² are independently C₁₋₁₂₀alkyl, preferably R¹ and R² are    independently selected from methyl, ethyl, propyl, and isopropyl;    and-   R⁵ is —OH, cyano, C₁₋₁₀alkoxy, amino, O-aralkyl, O-aralkyl,    —OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl, —OC(O)aryl, or a glycine    substituent, preferably OH;-   R⁶ is H or C₁₋₁₀alkyl;-   R⁷ is selected from halogen, —OH, C₀₋₆alkyl-COOR⁶, C₁₋₆alkyl,    C₁₋₆alkoxy, amino, amido, cyano, and nitro;-   m is an integer from 0 to 4, preferably 0.

In certain embodiments, W is (R)(R) and each occurrence of R isindependently H or a C₁₋₂alkyl. In certain such embodiments, eachoccurrence of R is H.

In certain embodiments, R⁵ is selected from cyano, C_(1-m)alkoxy, amino,O-aralkyl, —OC(O)C₁₋₁₀aralkyl, —OC(O)C_(1-m)alkyl, and —OC(O)aryl.

In certain embodiments X is S, W is O, R¹ and R² are independentlyselected from methyl, ethyl, propyl, and isopropyl, and R⁵ is OH. Incertain such embodiments, R¹ and R² are the same and are togetherselected from methyl, ethyl, propyl, and isopropyl. In certain suchembodiments, R¹ and R² are both methyl.

In certain embodiments, Z is N.

In certain embodiments, Z is CH.

In certain embodiments, a compound of formula (II) is selected from

In certain embodiments, a compound of formula (II) is

In other embodiments, the organic arsenicals are compounds having astructure of formula (III)

-   wherein-   X is S or Se, preferably S;-   W is O, S, or (R)(R), where each occurrence of R is independently H    or a C₁₋₂alkyl, preferably O;-   R¹ and R² are independently C₁₋₁₀alkyl, preferably R¹ and R² are    independently selected from methyl, ethyl, propyl, and isopropyl;    and-   R⁵ is —OH, cyano, C₁₋₁₀alkoxy, amino, O-aralkyl, O-aralkyl,    —OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl, —OC(O)aryl, or a glycine    substituent, preferably OH;-   R⁶ is H or C₁₋₁₀alkyl;-   R⁷ is selected from halogen, —OH, C₀₋₆alkyl-COOR⁶, C₁₋₆alkyl,    C₁₋₆alkoxy, amino, amido, cyano, and nitro;-   m is an integer from 0 to 4, preferably 0.

In certain embodiments, W is (R)(R) and each occurrence of R isindependently H or a C₁₋₂alkyl. In certain such embodiments, eachoccurrence of R is H.

In certain embodiments, R⁵ is selected from cyano, C₁₋₁₀alkoxy, amino,O-aralkyl, —OC(O)C₁₋₁₀aralkyl, —OC(O)C₁₋₁₀alkyl, and —OC(O)aryl.

In certain embodiments X is S, W is O, R¹ and R² are independentlyselected from methyl, ethyl, propyl, and isopropyl, and R⁵ is OH. Incertain such embodiments, R¹ and R² are the same and are togetherselected from methyl, ethyl, propyl, and isopropyl. In certain suchembodiments, R¹ and R² are both methyl.

In certain preferred embodiments, a compound of formula (II) has thefollowing structure

The invention further provides pharmaceutical compositions comprisingformula (I), formula (II), or formula (III), or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable diluent orcarrier. In certain embodiments, the pharmaceutical composition is anaqueous solution that has a pH greater than about 5, preferably in therange from about 5 to about 8, more preferably in the range from about 5to about 7.

Another aspect of the invention provides a method for the treatment ofcancer comprising administering a therapeutically effective amount of acompound of formula (I), formula (II), or formula (III).

The invention also relates to the use of a compound of formula (I),formula (II), or formula (III), or a pharmaceutically acceptable saltthereof, in the manufacture of a medicament for the treatment of cancer.

In certain embodiments, the cancer is selected from a solid tumor, suchas brain, lung, liver, spleen, kidney, lymph node, small intestine,pancreas, blood cells, bone, colon, stomach, breast, endometrium,prostate, testicle, ovary, central nervous system, skin, head and neck,esophagus, or bone marrow, or a hematological cancer, such as leukemia,acute promyelocytic leukemia, lymphoma, multiple myeloma,myelodysplasia, myeloproliferative disease, or refractory leukemia. Incertain such embodiments, the cancer is a leukemia selected from acuteand chronic leukemia.

In certain embodiments, the cancer is a lymphoma selected fromnon-Hodgkin's and Hodgkin's lymphoma. In certain embodiments, thenon-Hodgkin's lymphoma is selected from peripheral T-cell lymphoma(PTCL), diffuse large B-cell lymphoma, and marginal zone lymphoma. Incertain embodiments, the Hodgkin's lymphoma is Hodgkin's nodularsclerosis.

Thus, in another aspect, the invention comprises a method of treating apatient with cancer comprising administering to the patient acomposition comprising a compound of formula I, formula TI, or formulaIII, or pharmaceutical composition as described above. Thetherapeutically effective amount of a compound may be 0.1-1000 mg/kg,1-500 mg/kg, or 10-100 mg/kg. In particular embodiments, the method maycomprise administering the composition daily. It is further contemplatedthat treatment methods may involve multiple administrations. The methodmay comprise administering the compound daily such as by injection.Alternative routes and methods of administration described in thespecification may also be used and the mode of administration willmainly depend on the type and location of the cancer. In certainembodiments, the method further comprises administering one or moreadditional agents to the patient. The additional agent may beall-trans-retinoic acid, 9-cis retinoic acid, Am-80, or ascorbic acid.The use of other adjunct cancer therapies, such as chemotherapy,radiotherapy, gene therapy, hormone therapy, and other cancer therapiesknown in the art are also contemplated in conjunction with the methodsof the present invention.

Various methods of administration are contemplated, including regional,systemic, direct administration and by perfusion. Such methods includeadministration by injection, oral routes, intravenous, intraarterial,intratumoral, administration to tumoral vasculature, intraperitoneal,intratracheal, intramuscular, endoscopical, intralesional, percutaneous,subcutaneous, topical, nasal, buccal, mucosal, anogenital, rectal andthe like.

Definitions

The term “C_(x-y)alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from x to y carbons in the chain, includinghaloalkyl groups such as trifluoromethyl and 2,2,2-tirfluoroethyl, etc.C₀alkyl indicates a hydrogen where the group is in a terminal position,a bond if internal. The terms “C_(2-y)alkenyl” and “C_(2-y)alkynyl”refer to substituted or unsubstituted unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “C₁₋₆alkoxy” refers to an C₁₋₆alkyl group having an oxygenattached thereto. Representative alkoxy groups include methoxy, ethoxy,propoxy, tert-butoxy and the like. An “ether” is two hydrocarbonscovalently linked by an oxygen. Accordingly, the substituent of an alkylthat renders that alkyl an ether is or resembles an alkoxy.

The term “C₁₋₆aralkyl”, as used herein, refers to a C₁₋₆alkyl groupsubstituted with an aryl group.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon. The term “aryl” also includes polycyclicring systems having two or more cyclic rings in which two or morecarbons are common to two adjoining rings wherein at least one of therings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline,and the like.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include, for example, a halogen, ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

As used herein, the term “regimen” is a predetermined schedule of one ormore therapeutic agents for the treatment of a cancer. Accordingly, whena therapeutic agent is administered “alone,” the regimen does notinclude the use of another therapeutic agent for the treatment ofcancer.

In certain embodiments, the compound is administered daily for five daysevery four weeks. In certain embodiments, the compound is administeredonce daily for five days every four weeks, preferably for fiveconsecutive days. In certain alternative embodiments, the compound isadministered two days a week for three weeks, followed by one week off.In certain such embodiments, the compound is administered for twoconsecutive days or two non-consecutive days (e.g., with one, two,three, or even four days in between doses) a week for three weeks,followed by one week off. In certain embodiments, these protocols can berepeated indefinitely.

In certain embodiments, such dosing is by intravenous administration. Incertain alternative embodiments, such dosing is by oral administration.In certain such embodiments, the compound is administered intravenouslyat a dose of about 200-420 mg/m² or about 250 to 350 m/m². In certainembodiments, the compound is administered at a dose of about 200, about250, about 300, about 350, about 400 or even about 420 mg/m². In certainembodiments, the compound is administered orally at a total daily doseof 300 to about 700 mg or about 400 to about 600 mg. In certainembodiments, the compound is administered at a total daily dose of 300,about 400, about 500, about 600, or even about 700 mg.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

Toxicity of Inorganic Vs. Organic Arsenicals

The use of arsenic trioxide is limited by its toxicity. OA, on the otherhand, are much less toxic, to the extent that the methylation ofinorganic arsenic in vivo into OA has been considered to be adetoxification reaction. The OA monomethylarsinic acid anddimethylarsinic acid are the primary metabolites of inorganic arsenic(Hughes et al., 1998). Inorganic arsenicals, including arsenic trioxide,have varied effects on many organ systems, including cardiovascularsystem, gastrointestinal tract, kidneys, skin, nervous system, andblood. Inorganic arsenicals are particularly toxic to the liver, causinginfiltration, central necrosis, and cirrhosis (IARC, 1980: ACGIH, 1991;Beliles et al., 1994; Goyer et al., 1996). There is now sufficientevidence that inorganic arsenic compounds are skin and lung carcinogensin humans (Goyer et al., 1996).

The toxicity of a given arsenical is related to the rate of itsclearance from the body and to the extent of its tissue accumulation(Beliles et al., 1994). In general, toxicity increases in the followingsequence: organic arsenicals <As⁵⁺<As³⁺ (including arsenictrioxide)<arsine. Unlike inorganic arsenicals, no deaths or seriouscases of toxicity due to OA have been reported in the literature.Consequently, in mammals the methylation of inorganic arsenic has beenconsidered a detoxification mechanism because of the lower toxicity ofmethylated OA, and their fast excretion and low retention (Beliles etal., 1994; Goyer et al., 1996). A good example is that ofdimethylarsinic acid, an organic compound, the predominant urinarymetabolite excreted by most mammals after exposure to inorganic arsenic,including arsenic trioxide. In in vivo toxicity studies in mice, afterintraperitoneal administration of arsenic trioxide, the LD₅₀ (a dose atwhich 50% of animals die due to acute toxicity) was 10 mg/kg,(Investigator's Brochure, 1998), while after administration ofdimethylarsinic acid, the LD₅₀ was 500 mg/kg (MSDS, 1998).

Cancer Treatment

The organic arsenicals of the current invention may be used to treat avariety of cancers, including all solid tumors and all hematologicalcancers, including leukemia, lymphoma, multiple myeloma, myelodysplasia,or myeloproliferative disorders. The organic arsenical can also be usedto treat hematological cancers that have become refractory to otherforms of treatment.

In certain embodiments, the cancer is a lymphoma selected fromnon-Hodgkin's and Hodgkin's lymphoma. In certain embodiments, thenon-Hodgkin's lymphoma is selected from peripheral T-cell lymphoma(PTCL), diffuse large B-cell lymphoma, and marginal zone lymphoma. Incertain embodiments, the Hodgkin's lymphoma is Hodgkin's nodularsclerosis.

Lymphoma is a type of blood cancer that occurs when lymphocytes—whiteblood cells that help protect the body from infection and disease—beginbehaving abnormally. Abnormal lymphocytes may divide faster than normalcells or they may live longer than they are supposed to. Lymphoma maydevelop in many parts of the body, including the lymph nodes, spleen,bone marrow, blood, or other organs. There are two main types oflymphomas: Hodgkin lymphoma and non-Hodgkin lymphoma (NHL).

Peripheral T-cell lymphomas are tumors composed of mature T-cells (notB-cells). Peripheral T-cell lymphomas such as angioimmunoblastic T-celllymphoma or anaplastic large cell lymphoma can arise in lymph nodes,while others like subcutaneous panniculitis-like T-cell lymphoma, nasalNK/T-cell lymphoma, or intestinal T-cell lymphoma can arise inextranodal sites.

Large cell lymphomas are the most common type of lymphoma. These cancersmay arise in lymph nodes or in extranodal sites, including thegastrointestinal tract, testes, thyroid, skin, breast, central nervoussystem, or bone and may be localized or generalized (spread throughoutthe body).

Marginal zone tumors are indolent B-cell lymphomas and may occur eitheroutside lymph nodes (extranodal) or within lymph nodes (nodal). They aredivided into two categories depending on the location of the lymphoma.Mucosa-associated lymphoid tissue lymphomas (also called MALT orMALTomas) are forms of marginal zone lymphomas that affect placesoutside the lymph nodes (such as the gastrointestinal tract, eyes,thyroid, salivary glands, lungs, or skin). Nodal marginal zone B-celllymphomas are uncommon and are sometimes called monocytoid B-celllymphomas.

In Hodgkin's nodular sclerosis, the involved lymph nodes contain areascomposed of Reed-Sternberg cells mixed with normal white blood cells.The lymph nodes often contain prominent scar tissue, hence the namenodular sclerosis (scarring). This subtype is the most common, making up60% to 75% of all cases of Hodgkin's lymphoma.

Pharmaceutical Compositions

The preparation of a pharmaceutical composition that contains at leastone organic arsenical or additional active ingredient will be known tothose of skill in the art in light of the present disclosure, asexemplified by Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, incorporated herein by reference. Moreover, foranimal (e.g., human) administration, it will be understood thatpreparations should meet sterility, pyrogenicity, general safety andpurity standards as required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof; as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The organic arsenical may be combined with different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, injection, infusion,continuous infusion, localized perfusion bathing target cells directly,via a catheter, via a lavage, in lipid compositions (e.g., liposomes),or by other method or any combination of the forgoing as would be knownto one of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to a patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an organic arsenical compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 0.1 mg/kg/body weight, 0.5mg/kg/body weight, 1 mg/kg/body weight, about 5 mg/kg/body weight, about10 mg/kg/body weight, about 20 mg/kg/body weight, about 30 mg/kg/bodyweight, about 40 mg/kg/body weight, about 50 mg/kg/body weight, about 75mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/bodyweight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, about750 mg/kg/body weight, to about 1000 mg/kg/body weight or more peradministration, and any range derivable therein. In non-limitingexamples of a derivable range from the numbers listed herein, a range ofabout 10 mg/kg/body weight to about 100 mg/kg/body weight, etc., can beadministered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including, but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The organic arsenical may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts includethe salts formed with the free carboxyl groups derived from inorganicbases such as for example, sodium, potassium, ammonium, calcium orferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising, but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount of the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Thus, preferred compositionshave a pH greater than about 5, preferably from about 5 to about 8, morepreferably from about 5 to about 7. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

Combination Therapy

It is an aspect of this invention that the organic arsenical can be usedin combination with another agent or therapy method, preferably anothercancer treatment. The organic arsenical may precede or follow the otheragent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not elapse between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. For example, in suchinstances, it is contemplated that one may contact the cell, tissue ororganism with two, three, four or more modalities substantiallysimultaneously (i.e., within less than about a minute) with the organicarsenical. In other aspects, one or more agents may be administeredwithin about 1 minute, about 5 minutes, about 10 minutes, about 20minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours,about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours,about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours,to about 48 hours or more prior to and/or after administering theorganic arsenical. In certain other embodiments, an agent may beadministered within of from about 1 day, about 2 days, about 3 days,about 4 days, about 5 days, about 6 days, about 7 days, about 8 days,about 9 days, about 10 days, about 11 days, about 12 days, about 13days, about 14 days, about 15 days, about 16 days, about 17 days, about18 days, about 19 days, about 20, to about 21 days prior to and/or afteradministering the organic arsenical. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several weeks (e.g., about 1, about 2, about 3, about 4,about 5, about 6, about 7 or about 8 weeks or more) lapse between therespective administrations.

Various combinations may be employed, the organic arsenical is “A” andthe secondary agent, which can be any other therapeutic agent, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the therapeutic compositions of the present inventionto a patient will follow general protocols for the administration ofchemotherapeutics, taking into account the toxicity, if any. It isexpected that the treatment cycles would be repeated as necessary. Italso is contemplated that various standard therapies or adjunct cancertherapies, as well as surgical intervention, may be applied incombination with the described arsenical agent. These therapies includebut are not limited to chemotherapy, radiotherapy, immunotherapy, genetherapy and surgery. The section below describes some adjunct cancertherapies:

Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabine, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,or any analog or derivative variant of the foregoing.

Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and U V-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells. The terms “contacted” and “exposed,”when applied to a cell, are used herein to describe the process by whicha therapeutic construct and a chemotherapeutic or radiotherapeutic agentare delivered to a target cell or are placed in direct juxtapositionwith the target cell. To achieve cell killing or stasis, both agents aredelivered to a cell in a combined amount effective to kill the cell orprevent it from dividing.

Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionucleotide, ricin A chain, cholera toxin, pertussis toxin, etc.)and serve merely as a targeting agent. Alternatively, the effector maybe a lymphocyte carrying a surface molecule that interacts, eitherdirectly or indirectly, with a tumor cell target. Various effector cellsinclude cytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with gene therapy. The general approach for combined therapyis discussed below. Generally, the tumor cell must bear some marker thatis amenable to targeting, i.e., is not present on the majority of othercells. Many tumor markers exist and any of these may be suitable fortargeting in the context of the present invention. Common tumor markersinclude carcinoembryonic antigen, prostate specific antigen, urinarytumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

Gene Therapy

In yet another embodiment, the secondary treatment is a secondary genetherapy in which a therapeutic polynucleotide is administered before,after, or at the same time a first therapeutic agent. Delivery of thetherapeutic agent in conjunction with a vector encoding a gene productwill have a combined anti-hyperproliferative effect on target tissues.

Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies. Curative surgeryincludes resection in which all or part of cancerous tissue isphysically removed, excised, and/or destroyed. Tumor resection refers tophysical removal of at least part of a tumor. In addition to tumorresection, treatment by surgery includes laser surgery, cryosurgery,electrosurgery, and microscopically controlled surgery (Mohs' surgery).It is further contemplated that the present invention may be used inconjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art will, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Synthesis of S-dimethylarsino-thiosuccinic acid (MER1),S-dimethylarsino-salicylic acid (SAL1), and S-(dimethylarsino)glutathione (SGLU1)

MER-1: Mercaptosuccinic acid, 4.5 g, was placed in 100 mL of glyme(1,2-dimethoxyethane) in a 250 mL round-bottom flask. Four mL ofdimethylchloroarsine (0.03 mol) was added drop-wise, followed by 4 mL ofdiethylamine (0.04 mol), again dropwise. The reaction mixture wasstirred for 20 h at room temperature. A white precipitate ofdiethylamine hydrochloride was formed and was separated by filtration.The solution of MER1 in the glyme was greatly reduced in volume byevaporation at reduced pressure. White crystals of MER1 were separatedby filtration and washed with cold distilled water. The colorlesscrystalline product was then recrystallized from ethanol-water to aconstant melting point of 150° C.

SAL-1: In a 100 mL flask 5 g of 2-mercapto benzoic acid (thiosalicylicacid), 75 mL of glyme, 5 mL of dimethylchloroarsine, and 5 mLdiethylamine were placed. The mixture was refluxed for 1 hour under anatmosphere of nitrogen and stirred at room temperature overnight. Theprecipitate of diethylamine hydrochloride was separated by filtration.The filtrate was evaporated slowly under reduced pressure until crystalsof the product separate. The evaporated solution containing the productwas chilled in ice and the cold solution was filtered. Crystals of theproduct were recrystallized from ethanol to a constant melting point of97° C.

SGLU-1: Glutathione (14.0 g, 45.6 mmol) was stirred rapidly in glymewhile dimethylchoroarsine (6.5 g, 45.6 mmol) was added dropwise.Pyridine (6.9 g, 91.2 mmol) was then added to the slurry and the mixturewas subsequently heated to reflux. The heat was removed immediately andthe mixture stirred at room temperature for 4 h. Isolation of theresultant insoluble solid and recrystallization from ethanol afforded 4as the pyridine hydrochloride complex (75% yield): mp 115-118° C.; NMR(D₂O) δ1.35 (s, 6H), 1.9-4.1 (m's, 10H), 7.8-9.0 (m, 5H); mass spectrum(m/e) 140, 125, 110, 105, 79, 52, 45, 36. This material is not used forthe examples described herein, but has been used in biological assays asdescribed in Banks, C. H., et al. (J. Med. Chem. (1979) 22: 572-575),which is incorporated herein by reference in its entirety.

Example 2 Alternate Synthesis of S-Dimethylarsinoglutathione

The following procedure describes the manner of preparation ofS-dimethylarsinoglutathione. The quantities used can be multiplied ordivided with equal success if the respective ratios are maintained.

Dimethylchloroarsine.

Dimethylarsinic acid, (CH₃)₂As(O)OH was supplied by the Luxembourg

Chemical Co., Tel Aviv, Israel. The product was accompanied by astatement of its purity and was supplied as 99.7% pure. Thedimethylarsinic acid was dissolved in water-hydrochloric acid to pH 3. Astream of sulfur dioxide was passed through this solution for about onehour. Dimethylchloroarsine separated as a heavy, colorless oil. The twoliquid phases, water/(CH₃)₂AsCl were separated using a separatoryfunnel. The chlorodimethylarsine was extracted into diethylether and theether solution was dried over anhydrous sodium sulfate. The driedsolution was transferred to a distillation flask which was heated slowlyto evaporate the ether. The remaining liquid, dimethylchloroarsine waspurified by distillation. The fraction boiling at 106-109° C. wascollected. The product, a colorless oil, displays a simple ¹H NMRresonance at 1.65 ppm.

S-Dimethylarsinoglutathione

In a 500 mL flask, 7 g of glutathione was used as received from theAldrich Chemical Co., purity 98% and dissolved in 250 mL of1,2-dimethoxyethane. To this solution was added 3.3 g ofdimethylchloroarsine. This was followed by the addition of 3.5 g ofpyridine (redistilled after drying over NaOH pellets). The solution wasrefluxed for one hour after which time it was stirred at roomtemperature for three hours.

The desired product, S-dimethylarsinoglutathione was separated as thepyridine hydrochloride complex. The solid was removed by filtration andwashed thoroughly with 1,2-dimethoxyethane. It was subsequently driedover anhydrous calcium chloride in vacuo. The yield ofS-dimethylarsinoglutathione pyridine hydrochloride was 10.3 g and themelting point was 135-140° C. This material was used in the biologicalassays described above in examples 2 to 12.

Example 3 Pyridine Hydrochloride Free Synthesis ofS-dimethylarsinoglutathione (GLU)

Dimethylarsinoglutathione is made using an adapted of Chen (Chen, G. C.,et al. Carbohydrate Res. (1976) 50: 53-62) the contents of which arehereby incorporated by reference in their entirety. Briefly,dithiobis(dimethylarsinoglutamine) is dissolved in dichloromethane undernitrogen. Tetramethyldiarsine is added dropwise to the solution and thereaction is stirred overnight at room temperature under nitrogen andthen exposed to air for 1 h. The mixture is then evaporated to drynessand the residue is washed with water and dried to give a crude solidthat is recrystallized from methanol to giveS-dimethylarsinoglutathione.

Example 4 Third Synthesis of Pyridine Hydrochloride Free5-dimethylarsinoglutathione (GLU)

S-dimethylarsinoglutathione is made using the procedure of Cullen et al.(J. Inorg. Biochem. (1984) 21: 179-194) the contents of which are herebyincorporated by reference in their entirety. Briefly, dimethylarsinicacid and glutathione are dissolved in water under a nitrogen atmosphereand stirred. The resulting solution is stirred for 12 h and thenevaporated to dryness under reduced pressure without heating to give asolid that is extracted with cold methanol. The methanol solution isthen evaporated to dryness under reduced pressure and the resultingsolid is recrystallized from methanol/water, collected, and dried togive S-dimethylarsinoglutathione.

Example 5 Preparation of Dimethylchloroarsine

A 3 L, 3 necked round bottom flask was equipped with a mechanicalstirrer assembly, an additional funnel, thermometer, nitrogen inlet, anda drying tube was placed in a bath. The flask was charged with cacodylicacid (250 g) and concentrated HCl (825 mL) and stirred to dissolve.After the cacodylic acid was completely dissolved, the solution waswarmed to 40° C. To the stirring solution, hypophosphorous acid (H₃PO₂)(50% solution, 250 g) was added dropwise, maintaining the reactiontemperature between 40-50° C. After approximately 50 mL of H₃PO₂ hadbeen added, the solution became cloudy and the temperature of thereaction rose rapidly at which time an external cooling bath was used tomaintain the reaction temperature between 40-50° C. The addition of theH₃PO₂ was continued, maintaining the reaction temperature in the desiredrange. After the addition of H₃PO₂ was complete, the reaction was heldbetween 40-45° C. for 15 minutes while stirring. The external bath wasremoved and the stirring was continued. The reaction was allowed to stirand cool to <30° C. After the temperature of the reaction mixturedropped to 30° C. or less, methylene chloride (300 mL) was added and theresulting mixture was stirred to extract the product into the methylenechloride. Stirring was discontinued and the layers were allowed toseparate over ½ hour. The layers were separated and the methylenechloride layer was dried over anhydrous sodium sulfate with stirring fora minimum of 1 hour. The mixture may be allowed to sit under a nitrogenatmosphere for a maximum of 72 hours. The organic mixture was filteredto remove the sodium sulfate and the methylene chloride was removed byatmospheric distillation. The crude residual product was distilled undera nitrogen atmosphere, through an 8″ Vigreux or packed column. Theproduct fraction with bp 104-106° C. at atmospheric pressure wascollected.

Preparation of S-Dimethylarsinoglutathione

A 5 L, three necked round bottom flask was equipped with a mechanicalstirrer assembly, thermometer, addition funnel, nitrogen inlet, and adrying tube was placed in a cooling bath. A polyethylene crock wascharged with glutathione-reduced (200 g) and deionized water (2 L) andstirred under a nitrogen atmosphere to dissolve all solids. The mixturewas filtered to remove any insoluble material and the filtrate wastransferred to the 5 L flask. While stirring, ethanol, 200 proof (2 L)was added and the clear solution was cooled to 0-5° C. using anice/methanol bath. Pyridine (120 g) was added followed by a dropwiseaddition of Me₂AsCl (120 g) over a minimum of 1 hour. The reactionmixture was stirred at 0-5° C. for a minimum of 2 hours prior to removalof the cooling bath and allowing the mixture to warm to room temperatureunder a nitrogen atmosphere with stirring. The reaction mixture wasstirred overnight (>15 hrs) at room temperature under a nitrogenatmosphere at which time a white solid may precipitate. The reactionmixture was concentrated to a slurry (liquid and solid) at 35-45° C.using oil pump vacuum to provide a white solid residue. As much water aspossible is removed, followed by two coevaporations with ethanol toazeotrope the last traces of water. The white solid residue was slurriedin ethanol, 200 pf. (5 L) under a nitrogen atmosphere at roomtemperature overnight. The white solid was filtered and washed withethanol, 200 pf. (2×500 mL) followed by acetone, ACS (2×500 mL). Theresulting solid was transferred to drying trays and vacuum oven driedovernight at 25-35° C. using oil pump vacuum to provide pyridiniumhydrochloride-free S-dimethylarsinoglutathione as a white solid with amelting point of 189-190° C.

Preparation of Dosage Form of S-Dimethylarsinoglutathione

A solution of S-dimethylarsinoglutathione in water for injection (WFI)was adjusted to pH 5.0 to 5.5 with NaOH or HCl. The resulting solutionwas then filtered through a 0.2 micron Sartopore 2 filter and a Flexiconfilling unit was used to deliver 150 mg per Type 1 borosilicate glassvial (Wheaton). The filled vials were then lyophilized in a Hull 48Lyophilizer unit by first loading the vials on the shelf and ramping thetemperature to −40° C. at a cooling rate of 0.5° C. per minute. Theshelf temperature was then held at −40° C. for 300 minutes. A vacuum wasthen applied at 75 micron and the shelf temperature was ramped up to 5°C. at a rate of 0.1° C. per minute. The shelf temperature was then heldat 5° C. for 1,000 minutes before applying the vacuum at 50 micron. Theshelf temperature was then ramped up to 25° C. at a rate of 0.1° C. perminute and the temperature was held at 25° C. for 720 minutes. The shelftemperature was then reduced to 5° C. and held until the finalstoppering step, at which time the chamber was returned to 640,000 mmTorr with nitrogen and the vials were stoppered with gray butyllyophilization stoppers and finally crimped with aluminum seals toprovide S-dimethylarsinoglutathione as a white to off-white cake with amoisture content of 1.8%. The total time for the lyophilizationprocedure was 47 hours. The lyophilized S-dimethylarsinoglutathione wasthen reconstituted with 2.0 mL sterile water to provide a clear,colorless solution with a final concentration of 75±7.5 mgS-dimethylarsinoglutathione per mL and a pH of 4.5 to 6.0.

Example 6 Preparation of Dimethylchloroarsine (DMCA)

A 3-neck round-bottom flask (500 mL) equipped with mechanical stirrer,inlet for nitrogen, thermometer, and an ice bath was charged withcacodylic acid (33 g, 0.23 mol) and conc. hydrochloric acid (67 mL). Ina separate flask, a solution of SnCl₂.2H₂O (54 g, 0.239 mol) in conc.hydrochloric acid (10 mL) was prepared. The SnCl₂.2H₂O solution wasadded to the cacodylic acid in HCl solution under nitrogen whilemaintaining the temperature between 5° C. and 10° C. After the additionwas complete, the ice bath was removed and the reaction mixture wasstirred at ambient temperature for 1 h. The reaction mixture wastransferred to a separatory funnel and the upper layer (organic)collected. The bottom layer was extracted with dichloromethane (DCM)(2×25 mL). The combined organic extract was washed with 1 N HCl (2×10mL) and water (2×20 mL). The organic extract was dried over MgSO₄ andDCM was removed by rotary evaporation (bath temperature 80° C., undernitrogen, atmospheric pressure). The residue was further distilled undernitrogen. Two fractions of DMCA were collected. The first fractioncontained some DCM and the second fraction was of suitable quality (8.5g, 26% yield). The GC analysis confirmed the identity and purity of theproduct.

Preparation of S-Dimethylarsinoglutathione (SGLU-1)

A suspension of glutathione (18 g, 59 mmol) in a mixture ofwater/ethanol 1:1 v/v (180 mL) was cooled below 5° C. and under an inertatmosphere treated with triethylamine (10 mL, 74 mmol) in one portion.The mixture was cooled to 0-5° C. and DMCA (11 g, 78.6 mmol) was addeddropwise over a period of 10 min, while maintaining the temperaturebelow 5° C. The reaction mixture was stirred at 0-5° C. for 4 h, and theresulting solids were isolated by filtration. The product was washedwith ethanol (2×50 mL) and acetone (2×50 mL) and dried in vacuum at RTovernight, to give 11 g (46%) of SGLU-1. HPLC purity was 97.6% by area(average of 3 injections), Anal. Calcd. for C₁₂H₂₂AsN₃O₆S: C, 35.04; H,5.39; N, 10.12, S, 7.8. Found: C, 34.92; H, 5.31; N, 10.27, S, 7.68. ¹Hand ¹³C-NMR were consistent with the structure. The filtrate was dilutedwith acetone (150 mL) and placed in a refrigerator for 2 days. Anadditional 5.1 g (21%) of SGLU-1 was isolated as the second crop, HPLCpurity was 97.7% by area (average of 3 injections).

Preparation of S-Dimethylarsinoglutathione (SGLU-1)

In a 3 L three-neck flask equipped with a mechanic stirrer, droppingfunnel and thermometer under an inert atmosphere was prepared asuspension of glutathione (114.5 g, 0.37 mol) in a 1:1 (v/v) mixture ofwater/ethanol (1140 mL) and cooled to below 5° C. The mixture wastreated slowly (over 15 min) with triethylamine (63.6 mL, 0.46 mol)while maintaining the temperature below 20° C. The mixture was cooled to4° C. and stirred for 15 min and then the traces of undissolved materialremoved by filtration. The filtrate was transferred in a clean 3 Lthree-neck flask equipped with a mechanic stirrer, dropping funnel,nitrogen inlet, and thermometer and DMCA (70 g, 0.49 mol) (lot#543-07-01-44) was added slowly while maintaining the temperature at3-4° C. The reaction mixture was stirred at 1-4° C. for 4 h, and acetone(1.2 L) was added over a period of 1 h. The mixture was stirred for 90min between 2 and 3° C. and the resulting solid was isolated byfiltration. The product was washed with ethanol (2×250 mL) and acetone(2×250 mL) and the wet solids were suspended in ethanol 200 Proof (2000mL). The product was isolated by filtration, washed with ethanol (2×250mL) and acetone (2×250 mL) and dried in vacuum for 2 days at RT to give115 g (75%) of SGLU-1, HPLC purity >99.5% (in process testing).

Example 7 In Vitro Evaluation of Anti Cancer Activity of GMZ27

GMZ27, an organic arsine having the following structure

was tested in 72 hour MTS assays against different human acutemyelocytic leukemia (AML) cell lines and it was found that the IC₅₀ was0.56-0.86 μM. This activity was higher than the activity of arsenictrioxide against these cell lines (FIG. 27A). The anti-leukemic activityof GMZ27 was then evaluated in a long-term (7 day) colony-forming assay,where cells are grown in semi-solid medium. GMZ27 had significantlyhigher activity than arsenic trioxide against both human leukemia celllines and leukemic cells obtained from patients with acute or chronicleukemia (FIG. 27B).

The mechanisms of anti-cancer activity of GMZ27 and arsenic trioxidewere then compared. Arsenic trioxide (ATO) exerted its anti-leukemicactivity in cells other than APL via several mechanisms, includinginduction of apoptosis, alteration in the production of intracellularROS resulting in the modulation of cellular GSH redox system, celldifferentiation/maturation and possible effect on cell cycle regulation.

GMZ27 was more potent in induction of apoptosis than ATO. Results showthat it activated the mitochondrial apoptotic pathway, as it alteredmitochondrial membrane potential and cleaved caspase 9, but also byalternate, extrinsic, pathway since it cleaved caspase 8. This resultedin the induction of caspase 3 activity, cleavage of PARP, and binding ofannexin V to the cells (FIGS. 28 and 29).

Pretreatment of leukemic cells with buthionine sulfoximine (BSO) rendersthem more sensitive to GMZ27; while pretreatment with dthiothreitol(DTT) or N-acetylcysteine (NAC), which may increase intracellular GSH,rendered the cells less sensitive (FIG. 30). This suggested that GMZ27,like ATO, modulates the GSH redox system in leukemic cells, however, itdid so earlier and to a greater extent than ATO did (FIG. 31).

GMZ27, at low doses, was found to partially induce celldifferentiation/maturation as judged by the induction of CD11bmaturation marker on the surface of cells. This effect was marginalcompared with that of ATO (FIG. 32). GMZ27 had no effect on the cellcycle progression (FIG. 33).

Toxicity of GMZ7 against healthy donor peripheral blood mononuclearcells has been evaluated in a long-term colony forming assay. GMZ27 wasless toxic to normal cells than ATO (FIG. 34).

Studies to determine the toxicity of a single dose injection of GMZ27were performed in normal Swiss-Webster mice. Toxicity was measured onthe basis of mortality. It was found that the concentration of GMZ27that kills 50% of mice (LD₅₀) was 100 mg/kg. In contrast, the LD₅₀ forATO was much lower, at only 10 mg/kg.

Example 8 Preparation of N-(2-S-dimethylarsinothiopropionyl)glycine

N-(2-mercaptopropionyl)glycine (0.02 mol, 3.264 g) was placed in1,2-dimethoxyethane (50 mL) and dimethylchloroarsine (0.025 mol, 3.52 g)was added dropwise. The reaction mixture was stirred for 4 h at roomtemperature. A white precipitate of triethylamine hydrochloride salt wasthen separated by filtration and the solubtion was reduced in volume byevaporation at reduced pressure. The resulting residue was purified bycolumn chromatography to afford the desired product (3.5 g).

Example 9 Preparation of 2-(S-dimethylarsino)thionicotinic acid

2-Mercaptonicotinic acid (0.02 mol, 3 g) was placed in dichloromethane(50 mL) and dimethylchloroarsine (0.025 mol, 3.52 g) was added dropwise.The reaction was stirred at reflux for 4 h. The dichloromethane was thenremoved by distillation and the residue was dissolved in diethyl ether(50 mL) and washed with water (3×). The solution was dried over Na₂SO₄,filtered, and the desired product was obtained as a pale yellow solidafter concentration under reduced pressure.

Example 10 L-(+)-2-amino-3-(dimethylarsino)thio-3-methylbutanoic acid

L-(+)-2-amino-3-mercapto-3-methylbutanoic acid (0.01 mol, 1.55 g) wasplaced in dichloromethane (50 mL) and dimthylchloroarsine (0.015 mol,2.1 g) in dichlorormethane (5 mL) was added dropwise followed by thedropwise addition of triethylamine (1.6 g). The mixture was stirred for4 h and the desired product appeared as a floating white crystallinesolid after filtration of the reaction mixture. The crystalline solidwas washed with dichloromethane, ethyl acetate, and acetone sequentiallyto provide the desired product (1.6 g; mp 107-109° C.).

Example 11

A Phase II multi-center trial of SGLU-1 (darinaparsin) was conducted inpatients diagnosed with advanced lymphomas. Eligible patients requiredtherapy and received at least 1 prior therapy. Patients received 300mg/m² of darinaparsin intravenously for 5 consecutive days every 28 days(1 cycle) and were then evaluated for efficacy and safety by standardcriteria. Treatment continued until toxicity or progression. To date thestudy has accrued 22 patients (15 non Hodgkin's [NHL], 7 Hodgkin's); 12are male and 10 are female. Median age at baseline was 60.5 years(range: 28-80), ECOG performance status was <2, and median number ofprior therapies was 3 (range: 1-6). Thirteen subjects have received atleast 2 cycles of SGLU-1 and are evaluable for efficacy. Of these, 1(diagnosed with peripheral T-cell lymphoma (PTCL)) has achieved acomplete response (CR), 3 (diagnosed with diffuse large B-cell, marginalzone, and Hodgkin's nodular sclerosis, respectively) have achievedpartial responses (PRs), and 2 patients with NHL have achieved stabledisease (SD). In the patient with marginal zone lymphoma who achievedPR, no evidence for macroscopic disease was present, but microscopicdisease was detectable on random biopsies from normal appearing gastricmucosa. All responders had been heavily pretreated (PTCL: CHOP(cyclophosphamide, doxorubicin, vincristine, and prednisolone)×6, ICE(ifosfamide, carboplatin and etoposide)×1, and EPOCH (etoposide,vincristine, doxorubicin, cyclophosphamide, and prednisone)×2; diffuseB-cell: RCHOP (rituximab, cyclophosphamide, doxorubicin, vincristine,and prednisolone)×5, RICE (rituximab, ifosfamide, carboplatin andetoposide)×3, and radiation therapy; marginal zone: rituximab×8, RCVP(rituximab, cyclophosphamide, vincristine and prednisolone)×1, andgemcitabine×1; and Hodgkin's: ICE×1, CBV (cyclophosphamide, carmustineand etoposide)×1, gemcitabine+MDX-060 (Medarex)×6). A total of 49 cyclesof SGLU-1 have been administered. The only Grade 3 adverse event (AE)considered drug-related was wheezing. A total of 12 subjects havereported 37 serious adverse events (SAEs) while on study. Of these, only2 had SAEs that were considered drug-related (neutropenic fever, fall).In conclusion, SLGU-1 has been very well tolerated and has demonstratedpromising activity in heavily pretreated patients diagnosed withadvanced lymphoma. Initial responses (1 CR, 3 PRs, 2 SDs) have beenobserved among 13 evaluable patients.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims. Those skilled in the art will also recognizethat all combinations of embodiments described herein are within thescope of the invention.

All of the above-cited references and publications are herebyincorporated by reference.

1-20. (canceled)
 21. A method of treating a lymphoma selected fromdiffuse large B-cell lymphoma, marginal zone lymphoma, and Hodgkin'snodular sclerosis in a subject, the method comprising intravenouslyadministering a compound of Formula (I) or a pharmaceutically acceptablesalt thereof:

to thereby treat the subject, wherein a dose of the compound is 200 to420 mg/m².
 22. The method of claim 21, wherein the lymphoma comprisesrelapsed or refractory lymphoma.
 23. The method of claim 21, wherein thedose of the compound is 300 mg/m².
 24. The method of claim 23, whereinthe composition is administered daily for five consecutive days.
 25. Themethod of claim 24, wherein the composition is administered daily forfive consecutive days every 3-4 weeks.